This invention relates generally to apparatus for rapid control of the temperature of a liquid sample. More specifically, the present invention relates to thermal cycling apparatus for precisely controlling and rapidly varying the temperature of a sample repeatedly through a predetermined temperatures cycle.
In numerous areas of industry, technology, and research there is a need to reliably and reproducibly subject relatively small samples to thermal cycling. The need to subject a sample to repeated thermal cycles is particularly acute in biotechnology applications. In the biotechnology field, it is often desirable to repeatedly heat and cool small samples of materials over a short period of time. One such biological process that is regularly carried out is cyclic DNA amplification.
Cyclic DNA amplification, using a thermostable DNA polymerase, allows automated amplification of primer specific DNA, widely known as the xe2x80x9cpolymerase chain reaction.xe2x80x9d Automation of this process requires controlled and precise thermal cycling of reaction mixtures usually contained in a plurality of containers. In the past, the container of preference has been a standard, plastic microfuge tube.
Commercial programmable metal heat blocks have been used in the past to effect the temperature cycling of samples in microfuge tubes through the desired temperature versus time profile. However, the inability to quickly and accurately adjust the temperature of the heat blocks through a large temperature range over a short time period, has rendered the use of heat block type devices undesirable as a heat control system when carrying out the polymerase chain reaction.
Moreover, the microfuge tubes which are generally used have disadvantages. The material of the microfuge tubes, their wall thickness, and the geometry of microfuge tubes is a hinderance to rapid heating and cooling of the sample contained therein. The plastic material and the thickness of the wall of microfuge tubes act as an insulator between the sample contained therein and the surrounding medium thus hindering transfer of thermal energy. Also, the geometry of the microfuge tube presents a small surface area to whatever medium is being used to transfer thermal energy. The continued use of microfuge tubes in the art, with their suboptimal geometry, indicates that the benefits of improved thermal transfer (which come by increasing the surface area of a sample container for a sample of constant volume) has heretofore not been recognized.
Furthermore, devices using water baths with fluidic switching, (or mechanical transfer) have also been used as a thermal cycler for the polymerase chain reaction. Although water baths have been used in cycling a polymerase chain reaction mixture through a desired temperature versus time profile necessary for the reaction to take place, the high thermal mass of the water (and the low thermal conductivity of plastic microfuge tubes), has been significantly limiting as far as performance of the apparatus and the yields of the reaction are concerned.
Devices using water baths are limited in their performance. This is because the water""s thermal mass significantly restricts the maximum temperature versus time gradient which can be achieved thereby. Also, the water bath apparatus has been found to be very cumbersome due to the size and number of water carrying hoses and external temperature controlling devices for the water. Further the need for excessive periodic maintenance and inspection of the water fittings for the purpose of detecting leaks in a water bath apparatus is tedious and time consuming. Finally, it is difficult with the water bath apparatus to control the temperature in the sample tubes with the desired accuracy.
U.S. Pat. No. 3,616,264 to Ray shows a thermal forced air apparatus for cycling air to heat or cool biological samples to a constant temperature. Although the Ray device is somewhat effective in maintaining a constant temperature within an air chamber, it does not address the need for rapidly adjusting the temperature in a cyclical manner according to a temperature versus time profile such as is required for biological procedures such as the polymerase chain reaction.
U.S. Pat. No. 4,420,679 to Howe and U.S. Pat. No. 4,286,456 to Sisti et al. both disclose gas chromatographic ovens. The devices disclosed in the Howe and Sisti et al. patents are suited for carrying out gas chromatography procedures but do not provide thermal cycling which is substantially any more rapid than that provided by any of the earlier described devices. Rapid thermal cycling, while potentially useful for many procedures, is particularly advantageous for carrying out the polymerase chain reaction. Devices such as those described in the Howe and Sisti et al. patents are not suitable for efficiently and rapidly carrying out such reactions.
In view of the above described state of the art, the present invention seeks to realize the following objects and advantages.
It is an object of the present invention to provide an apparatus for accurately controlling the temperature of biological samples.
It is a further object of the present invention to provide a thermal cycling apparatus for quickly and accurately varying the temperature of biological samples according to a predetermined temperature versus time profile.
It is another object of the present invention to provide an apparatus suitable for subjecting a number of different biological samples to rapid thermal cycling.
It is also an object of the present invention to provide a thermal cycling apparatus having a thermal transfer medium of low thermal mass which can effectively subject samples to a large temperature gradient over a very short period of time.
It is a further object of the present invention to provide an apparatus which can subject a biological sample to rapid thermal cycling using air as a thermal transfer medium.
It is another object of the present invention to provide a thermal cycling apparatus which will heat samples located in a fluid chamber therein, by means of an internal heater, and will subsequently cool the samples by moving ambient fluid into the chamber, at the proper time in the thermal cycle, to cool the samples.
These and other objects and advantages of the invention will become more fully apparent from the description and claims which follow, or may be learned by the practice of the invention.
The present invention is an apparatus particularly suited for subjecting biological samples to rapid thermal cycling in order to carry out one or more of a number of procedures or processes. In one of its preferred forms, the apparatus includes a means for holding a biological sample. The means for holding a biological sample, or a sample chamber, is provided with an insulation means for retaining thermal energy and also a means for heating the interior of the sample chamber. Preferably, a high wattage incandescent lamp functions as a means for heating the interior of the sample chamber. A thermal insulator lines the interior of the sample chamber and functions to retain the heat generated by the lamp within the sample chamber and serves as an insulation means.
In order to rapidly cool the sample chamber, the preferred apparatus includes a means for forcing air into the sample chamber and a means for dispersing the air forced into the sample chamber. A high velocity fan functions to force air into the sample chamber and a rotating paddle functions to disperse the air into the chamber. A means for venting allows the air to escape from the sample chamber taking the unwanted heat with it. The present invention allows heating and cooling of a sample to take place both quickly and uniformly.
The apparatus of the present invention includes a control means for operating the apparatus through the desired time versus temperature profile. The present invention is particularly well suited for carrying out automated polymerase chain reactions.
Other embodiments of the present invention include a closed loop hot fluid compartment and a reaction compartment. The reaction compartment is located within the hot fluid compartment and can be accessed through a venting door for allowing the insertion of samples in capillary tubes which may contain a reaction mixture for the polymerase chain reaction. A heating coil is also located in the compartment and is regulated by a programmable set-point process controller via a thermocouple sensor which is also located in the compartment at a position directly adjacent the reaction compartment.
The heat coil is located up-stream of the reaction compartment, and a fan is located up-stream of the heat coil, such that, fluid blown across the heat coil by the blower unit passes through the reaction compartment and into the intake side of the blower unit in closed loop fashion. Baffles may be located between the heat coil and the reaction compartment in order to cause uniform homogenous mixing of the heated fluid before it passes through the reaction compartment.
Alternately, the fan may be placed downstream of the heating coil but before the reaction compartment. In this case, the fan blades serve as baffles to mix the heated fluid. At the correct time in the predetermined thermal cycle, the controller activates a solenoid that opens the venting door which vents fluid out of the compartment and allows cool (ambient) fluid to enter and cool the samples.
The controller of the present invention allows the chamber, and subsequently the samples located in the sample compartment therein, to pass through a predetermined temperature cycle corresponding to the denaturation, annealing and elongation steps in the polymerase chain reaction. In use, the apparatus of the present invention allows rapid optimization of denaturation, annealing, and elongation steps in terms of time and temperature, and shortened time periods (ramp times) between the temperatures at each step.
The present invention particularly decreases the total time required for completion of polymerase chain reaction cycling over prior art thermal cycling devices while at the same time significantly increasing specificity and yield.